Pretreatment with human serum butyrylcholinesterase alone prevents cardiac abnormalities, seizures, and death in Göttingen minipigs exposed to sarin vapor
Graphical abstract
Pretreatment with 7.5 mg/kg of Hu BChE alone protected Göttingen minipigs from GB vapor-induced cardiac abnormalities, seizures, and death by sequestering GB in blood.
Introduction
Organophosphorus (OP) nerve agents such as soman (GD), sarin (GB), VX, and tabun (GA), exert their toxicity by inhibiting acetylcholinesterase (AChE) in the central nervous system (CNS) [1], [2]. Exposure to OP agents results in the accumulation of acetylcholine, hyperstimulation of central and peripheral cholinergic receptors, and the resultant production and progression of toxic signs culminating with the development of generalized tonic–clonic seizures, cardiorespiratory collapse, and death [3], [4], [5], [6]. A novel approach for counteracting OP toxicity employs the use of an enzyme bioscavenger to sequester and neutralize these compounds before they reach their physiological targets [7]. Of the enzymes tested, human serum butyrylcholinesterase (Hu BChE; EC 3.1.1.8) appears to be most appropriate for human use [8]. A dose of 200 mg of Hu BChE is envisioned as a prophylactic treatment in humans that can protect from an exposure of up to 2 × LD50 of GD [9]. In addition to its use as a pretreatment for a variety of wartime scenarios, it also has potential use as a pretreatment for first responders reacting to intentional/accidental nerve gas release and as a post-exposure therapy for pesticide overexposure, cocaine overdose, or succinylcholine-induced apnea [8].
For ethical reasons, the efficacy of Hu BChE cannot be investigated in humans. Therefore, the effectiveness of Hu BChE against multiple LD50's of OP nerve agents was evaluated using different animal species, including rodents and non-human primates. It was shown that pretreatment with Hu BChE alone protected mice from toxicity due to GD, GB, VX, and GA [10], [11]. In addition to enhancing survivability, pretreatment with Hu BChE prevented the development of GD-induced cognitive impairments in rats [12]. Subsequent studies in rhesus monkeys showed that although a molar ratio of Hu BChE:OP of ∼1.2 was required to protect against 2.1 × LD50 of VX, a smaller ratio of 0.62 was sufficient against 3.3 × LD50 of GD [13]. As in rats, the enzyme also protected rhesus monkeys against GD-induced behavioral deficits, which were evaluated using a spatial discrimination task. More recently, the efficacy of Hu BChE was demonstrated in guinea pigs against cumulative s.c. challenges of up to 5.5 × LD50 of GD or 8 × LD50 of VX [14], [15]. No signs of OP poisoning were observed and all animals survived the duration of challenge. In non-human primates, four of six cynomolgus monkeys were protected against a cumulative challenge of 5.5 × LD50 of GD. The four surviving animals did not display any immediate or delayed signs of OP toxicity as revealed by examinations of blood chemistry and hematology parameters over 14 months [16].
Most efficacy studies described to date were conducted using i.v. or s.c. challenge of OP nerve agents. The pharmacokinetics, onset time, and severity of toxic manifestations following exposure to OPs depend not only on the animal species but also on the route of entry of OP agents. Since, inhalation is the most likely route of exposure to G-type nerve agents on the battlefield or in public places, the efficacy of Hu BChE should be evaluated against a vapor challenge. The first such study was described by Allon et al. [17], who reported that exogenously administered Hu BChE was effective in protecting guinea pigs from inhalation toxicity from nose-only exposure to GD. In the present study, we investigated for the first time the efficacy of Hu BChE as a prophylactic measure against whole-body exposure to a lethal dose of GB vapor. GB is a colorless and odorless liquid at room temperature, and can be hazardous in liquid as well as vapor form due to its relatively low vapor pressure [18]. This study was conducted in the Göttingen minipig, which is widely accepted as a surrogate for cardiorespiratory physiology in man, and is an attractive animal model for investigating neurotoxicology. Animals were pretreated with Hu BChE by i.m. injection, 24 h prior to whole-body exposure to 4.1 mg/m3 for 60 min of GB vapor (2.4 × LCt50). This value is equivalent to an LCt99. Pharmacokinetic studies reported previously [19] showed that Hu BChE delivered by i.m. injection attained peak activity in minipig blood at ∼24 h. Electrocardiographic (ECG) and electroencephalographic (EEG) recordings and pupil size were monitored throughout exposure. Blood drawn from a surgically implanted jugular vein catheter before and throughout the exposure period was analyzed for blood gases, electrolytes, metabolites, AChE and BChE activities, and the amount of GB present in plasma and red blood cells (RBCs). Results demonstrate that prophylaxis with a dose of 7.5 mg/kg of Hu BChE alone was effective in preventing cardiac abnormalities, neural toxicity, and death due to respiratory failure in minipigs exposed to a lethal dose of GB vapor.
Section snippets
Materials and methods
All animal studies were conducted in compliance with the Animal Welfare Act and other federal statutes and regulations stated in the Guide for the Care and Use of Laboratory Animals (NRC Publication, 1996 edition). All procedures with animals received prior approval from the Edgewood Chemical and Biological Center (ECBC) Institutional Animal Care and Use Committee and were performed in facilities fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care,
Circulating cholinesterase activity following exposure of minipigs to sarin vapor
Minipigs were pretreated with saline, 3.0, or 7.5 mg/kg of Hu BChE by i.m. injection and challenged with air or GB vapor 18–20 h later. However, due to an error in preparing the enzyme solution, two animals received 6.5 mg/kg of Hu BChE instead of 7.5 mg/kg. The average baseline AChE and BChE activities in the blood of all animals were 2.54 ± 0.06 and 0.16 ± 0.03 U/ml, respectively. As expected, circulating blood BChE activity reached 14.8 ± 2.1, 35.1 ± 1.9, and 41.2 ± 0.9 U/ml at 18–20 h following the
Discussion
Due to the inability of current antidotal regimens to provide complete protection against OP nerve agent toxicity, several alternatives such as pyridostigmine bromide and enzyme bioscavengers are being considered as prophylactic countermeasures [7]. Of the enzymes evaluated, Hu BChE appears to be most suitable for human use and is currently under advanced development. Hu BChE has been previously shown to be safe, to exhibit favorable pharmacokinetics, and to effectively protect rodents, guinea
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgements
The authors wish to thank Ms. Amy Michels for her assistance in preparation of the manuscript. This research was supported by funding from the Defense Threat Reduction Agency. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Army or the Department of Defense.
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- 1
Present address: Division of Bacterial & Rickettsial Diseases, Walter Reed Army Institute of Research, Silver Spring, MD 20910, United States.
- 2
Present address: Department of Neuroscience, Merck Research Laboratories, Kenilworth, NJ 07033, United States.
- 3
Present address: Closed Head Injury Branch, Center for Military Psychiatry and Neuroscience, Walter Reed Army Institute of Research, Silver Spring, MD 20910, United States.